Amyotrophic lateral sclerosis (ALS) is a fatal adult-onset neurodegenerative disease characterized by a selective loss of motor neurons. Mutations in SOD1 gene are the most frequent causes of familial ALS. Cystatin C (CysC), an endogenous cysteine protease inhibitor, is a major protein component of Bunina bodies in sporadic ALS and decreased in the patients' cerebrospinal fluid. Despite the prominent deposition of CysC in ALS, the role of CysC had not been clarified. In this study, we examined the potential neuroprotective activity of CysC against mutant SOD1-mediated toxicity. We found that exogenously added recombinant CysC protected neuronal cells including primary cultured motor neurons. The neuroprotective activity of CysC was dependent on coordinated activation of two distinct neuroprotective pathways: induction of autophagy through AMP-activated kinase and inhibition of aberrantly activated cathepsin B. Moreover, CysC, which was transduced into lysosomes under normal conditions, was leaked into cytosol and aggregated under oxidative stress conditions, implying the relationship between the neuroprotective activity of CysC and Bunina body formation. These data suggest that CysC is an endogenous neuroprotective agent and might be a novel promising therapeutic target for ALS.